Nfat5 is involved in the hyperosmotic regulation of Tmem184b: a putative modulator of ibuprofen transport in renal MDCK I cells.

Nuclear factor of activated T cells 5 (NFAT5) is a transcription factor involved in the regulation of several genes involved in the response to extracellular hyperosmolality. Recently, the uptake of ibuprofen by an as yet unknown carrier was suggested in Madin-Darby canine kidney (MDCK) I cells exposed to hyperosmolality. We therefore speculated that Nfat5 could be involved in the regulation of this ibuprofen carrier. Reverse transfection with siRNA against Nfat5 was used to knock down Nfat5 in MDCK I cells. The uptake of both radiolabelled taurine and ibuprofen was measured in MDCK I cells, first treated with siRNA against Nfat5 and afterwards cultivated with raffinose-supplemented normal growth medium (500 mOsm) for 24 h. The siRNA transfection resulted in knockdown of Nfat5, and uptake of both taurine and ibuprofen was significantly decreased in transfected MDCK I cells. The decrease in ibuprofen uptake indicates that Nfat5 is involved in upregulation of the ibuprofen carrier. A transcriptome analysis of MDCK I cells treated with siRNA against Nfat5 revealed 989 genes upregulated by Nfat5 during hyperosmotic exposure. From these genes, the gene product transmembrane protein 184b was found to be regulated by Nfat5, and Tmem184b was the only potential gene product involved in the uptake of ibuprofen in MDCK I cells. DATASET: The RNA sequencing dataset is available from the NCBI Gene Expression 452 Omnibus (https://www.ncbi.nlm.nih.gov/geo/) with the accession number GSE122074.

Transcriptome analysis identifies activated signaling pathways and regulated ABC transporters and solute carriers after hyperosmotic stress in renal MDCK I cells.

Hyperosmolality is found under physiological conditions in the kidneys, whereas hyperosmolality in other tissues may be associated with pathological conditions. In such tissues an association between inflammation and hyperosmolality has been suggested. During hyperosmotic stress, an important phenomenon is upregulation of solute carriers (SLCs). We hypothesize that hyperosmolality affects the expression of many SLCs as well as ABC transporters. Through RNA-sequencing and topological pathway analysis, the cell cycle, the cytokine-cytokine receptor interaction pathway, and the chemokine-signaling pathway were significantly activated in MDCK I cells after hyperosmotic treatment (Δ200 mOsm) with raffinose or NaCl. 9065, 8052 and 5018 genes were significantly regulated by raffinose, NaCl or urea supplementation (500 mOsm), respectively, compared to control (300 mOsm). Cytokines, that have not previously been associated with hyperosmolality, were identified. We further provide an overview of transport proteins that could be of relevance in tissues exposed to hyperosmolality. Especially Slc5a8 was found highly up-regulated.

Interaction of GABA-mimetics with the taurine transporter (TauT, Slc6a6) in hyperosmotic treated Caco-2, LLC-PK1 and rat renal SKPT cells.

The aim of the present study was to investigate if basic GABA-mimetics interact with the taurine transporter (TauT, Slc6a6), and to find a suitable cell based model that is robust towards extracellular changes in osmolality during uptake studies. Taurine uptake was measured in human Caco-2 cells, porcine LLC-PK1 cells, and rat SKPT cells using radiolabelled taurine. Hyperosmotic conditions were obtained by incubation with raffinose (final osmolality of 500mOsm) for 24h prior to the uptake experiments. Expression of the taurine transporter, TauT, was investigated at the mRNA level by real-time PCR. Uptake of the GABA-mimetics gaboxadol and vigabatrin was investigated in SKPT cells, and quantified by liquid scintillation or HPLC-MS/MS analysis, respectively. The uptake rate of [(3)H]-taurine was Na(+) and Cl(-) and concentration dependent with taurine with an apparent Vmax of 6.3±1.6pmolcm(-2)min(-1) and a Km of 24.9±15.0µM. ß-alanine, nipecotic acid, gaboxadol, GABA, vigabatrin, δ-ALA and guvacine inhibited the taurine uptake rate in a concentration dependent manner. The order of affinity for TauT was ß-alanine>GABA>nipecotic acid>guvacine>δ-ALA>vigabatrin>gaboxadol with IC50-values of 0.04, 1.07, 2.02, 4.19, 4.94, 31.4 and 39.9mM, respectively. In conclusion, GABA mimetics inhibited taurine uptake in hyperosmotic rat renal SKPT cells. SKPT cells, which seem to be a useful model for investigating taurine transport in the short-term presence of high concentrations of osmolytes. Furthermore, analogues of ß-alanine appear to have higher affinities for TauT than GABA-analogues.